Therapeutic uses of allogeneic myeloid progenitor cells

ABSTRACT

Myeloid function is enhanced by transplantation or infusion of allogeneic myeloid progenitor cells, including CMP, GMP, MEP and MKP cell subsets. Myeloid progenitors ameliorate sequelae of anemia and thrombocytopenia, and can prevent or treat gastrointestinal mucositis associated with chemotherapy, radiotherapy, and the like. The transplantation or infusion may be performed in the absence of HLA typing, and the cells may be mismatched at one or more Class I HLA loci. The transplantation may provide for treatment of ongoing disease, or prevention of disease in high risk patients.

BACKGROUND OF THE INVENTION

Blood cells are derived from hematopoietic stem and progenitor cells inthe bone marrow. During the process of differentiation, a pluripotentialstem cell gives rise to progenitor and effector cells that have a morelimited developmental repertoire, and which may give rise only to cellswithin a particular lineage. The common myeloid progenitor cell is theprecursor of megakaryocytes, erythrocytes, granulocytes, macrophages,dendritic cells, and mast cells. These cells comprise the innate immunesystem, which is involved in antigen presentation, phagocytosis, andother non-antigen specific responses.

Macrophages are one of the three types of phagocytic cells in the immunesystem. They are the mature form of monocytes, which circulate in theblood and differentiate continuously into macrophages upon migrationinto the tissues. Dendritic cells are also phagocytic, and arespecialized to take up antigen and display it for recognition bylymphocytes. Mast cells also differentiate in the tissues. They mainlyreside near small blood vessels and, when activated, release substancesthat affect vascular permeability. Although best known for their role inorchestrating allergic responses, they are believed to play a part inprotecting mucosal surfaces against pathogens.

There are three types of granulocyte, all of which are relatively shortlived and are produced in increased numbers during immune responses,when they leave the blood to migrate to sites of infection orinflammation. Neutrophils, which are the third phagocytic cell of theimmune system, are the most numerous and most important cellularcomponent of the innate immune response: hereditary deficiencies inneutrophil function lead to overwhelming bacterial infection, which isfatal if untreated. Eosinophils are thought to be important chiefly indefense against parasitic infections, because their numbers increaseduring a parasitic infection. The function of basophils is probablysimilar and complementary to that of eosinophils and mast cells.

Patients suffering from various diseases and therapies may have adeficiency on one or more of these myeloid lineage cells, whichdeficiency can result in increased susceptibility to bacterial andfungal infections. Leukopenia is usually characterized by a reducednumber of blood neutrophils, although a reduced number of lymphocytes,monocytes, eosinophils, or basophils may also contribute to thedecreased total cell count. Neutropenia accompanied by monocytopenia andlymphocytopenia is often a more serious disorder than neutropenia alone.Thrombocytopenia can also be a problem for myelosuppressed patients,stemming from failed megakaryocyte production. Severe thrombocytopeniaresults in a typical pattern of bleeding. Platelet transfusions can beused, but with discretion, because they may lose their effectivenesswith repeated use owing to the development of platelet alloantibodies.

For example, patients undergoing hematopoietic cell transplantation(HCT) receive myeloablative doses of chemo-radiation therapy that leadto depletion of hematopoietic stem cells (HSC), progenitor cells andmature cells, thus leading to a phase of treatment related pancytopenia.The reconstitution of a functional immune system after HCT is dependentupon the de novo regeneration of all hematopoietic lineages from HSC andprogenitor cells and on the function of mature cells contained in thegraft. Infections after HCT typically follow a reproducible time patterncorrelating with the kinetics of immune reconstitution, and despiteaggressive treatment, the mortality rate of infections in the absence ofimmune reconstitution can be very high.

Drugs are one of the most common causes of neutropenia. Drug-inducedneutropenia has several underlying mechanisms (immune, toxic,idiosyncratic, or hypersensitivity reactions), including severeneutropenia that predictably occurs after large doses of cytoreductivecancer drugs or radiotherapy and from that caused by viral infections.Cytotoxic chemotherapy induces neutropenia because of the highproliferative rate of neutrophil precursors and the rapid turnover ofblood neutrophils. Impaired neutrophil production can also occur whenleukemia, myeloma, lymphoma, or metastatic solid tumors infiltrate andreplace the bone marrow. Tumor-induced myelofibrosis may furtherextenuate neutropenia. Myelofibrosis can also occur from granulomatousinfections, Gaucher's disease, and radiotherapy.

Patients whose neutropenia is secondary to acquired disorders ofproduction arising from cancer or from chemotherapy are more likely todevelop serious bacterial or fungal infections because their overallimmune system is compromised. The integrity of the skin and mucousmembranes, the vascular supply to tissue, and the nutritional status ofthe patient also influence the risk of infections in acute neutropenia.Patients may also suffer from genetic or primary deficiencies of myeloidcells and are highly susceptible to infection as is seen, for example,in children with chronic granulomatous disease.

The treatment and prevention of infections, particularly in patientssuffering or at risk of myeloid cell deficiencies, are of great medicalconcern. The present invention addresses these issues.

RELEVANT LITERATURE

U.S. Pat. Nos. 6,465,247 and 6,761,883, herein specifically incorporatedby reference, characterize mammalian myeloid progenitor cells.Bitmansour et al (2002) Blood 100(13):4660-7 cotransplant congeniccommon myeloid progenitors (CMP) and granulocyte-monocyte progenitors(GMP) with a graft containing hematopoietic stem cells to enhancereconstitution of a tissue myeloid pool for protection against lethalchallenge with fungal and bacterial pathogens. Arber et al. (2003) Blood102:3504 provides an abstract relating to engraftment and protectionwith MHC-mismatched committed myeloid progenitors.

SUMMARY OF THE INVENTION

Compositions and methods are provided for enhancement of myeloidfunction in an individual, through transplantation or infusion ofallogeneic or xenogeneic myeloid progenitor cells. The transplantationor infusion of these progenitor cells provides for treatment of ongoingdisease or infection, or prevention of disease or infection, e.g. inhigh risk patients. It is also shown that transplantation or infusion ofthese progenitor cells provides for the treatment or prevention ofradiation-injured mucosa, e.g. in gastrointestinal mucositis, and thelike, which can be the result of chemotherapy, radiation therapy, andthe like.

Individuals that benefit from the methods of the invention includeimmunocompromised patients having a deficiency in myeloid or erythroidcell function, e.g. following myeloablative doses of chemotherapy orradiation therapy; patients suffering from neutropenia; patientssuffering from thrombocytopenia; patients suffering from chronicdisorders such as chronic granulomatous disease; sickle cell disease;and the like.

The myeloid progenitor cells comprise one or more of: common myeloidprogenitor cells (CMP); and the committed myeloid progenitors:erythroid/megakaryocytic progenitor (MEP), granulocyte/monocyteprogenitors (GMP); and megakaryocyte progenitor (MKP). Generally themyeloid progenitors are initially purified, and may be provided to thepatient in a purified form, or reconstituted as a mixture of cells, e.g.combined with mature platelets, red blood cells, stem cells, etc. Thecells are administered to the individual in a biologically effectivedose, e.g. by intravenous injection, etc. The myeloid progenitor cellswill usually have one or more Class I HLA mismatches relative to therecipient, and administration may be performed without HLA testing ofthe recipient, although in certain embodiments of the invention themyeloid progenitor cells may be HLA matched.

The cell therapy of the invention may be combined with administration ofcells, including hematopoietic stem cells; and may be combined withadministration of cytokines, particularly cytokines that stimulategrowth or enhance function of myeloid or erythroid lineage cells, e.g.G-CSF; GM-CSF; thrombopoietin; erythropoietin; and the like. The celltherapy may also be combined with agents including antifungal agents;antibiotics; anti-parasitic agents; and the like, as appropriate. Insome embodiments of the invention, the myeloid progenitor cells of theinvention are co-formulated with such cytokines and/or agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Engraftment of CMP/GMP with different donor-host antigendisparities. Absolute counts of CMP/GMP-derived splenocytes (A) orpercentages of CMP/GMP-derived bone marrow cells on day 7post-transplantation after transplantation of 10⁴ CMP and 2×10⁴ GMP incongenic, MHC-matched or MHC-mismatched hosts. Granulocytes (Mac-1⁺Gr-1⁺cells) are shown in black. CMP/CMP gave rise to cells of themyelomonocytic, erythroid and dendritic cell lineages as shown in spleenor bone marrow (B) with a similar distribution in the three donor-hostmouse strain combinations tested. CMP/GMP-engraftment was significantlyless in MHC-mismatched hosts as compared to congenic or MHC-matchedhosts. Group 1 vs group 3 *P=0.012, group 2 vs group 3 **P=0.02. Designof Experiment shown in FIG. 1C.

FIG. 2A-2B. Protection against Aspergillus fumigatus infection. (A)MHC-matched model (Table 1 group 4). HSC were harvested fromC57BL/Ka-Thy1.1 (H2^(b)) and CMP/GMP from C57BL/6-Ly5.2 (H2^(b)) mice.Mice were transplanted with 500 HSC alone (◯, n=10, 40% survival) or 500HSC and 10⁴ CMP and 2×10⁴ GMP (, n=10, 90% survival). *P=0.02 ( versus◯). (B) MHC-mismatched model (Table 1 group 5) HSC are fromB10.D2.Thy1.1 (H2^(d)) and CMP/GMP from C57BL/6-Ly5.2 (H2^(b)). Micewere transplanted with 500 HSC alone (◯, n=24, 33% survival) or with 500HSC and 10⁴ CMP and 2×10⁴ GMP (, n=25, 88% survival) or with 500 HSCand 4×10⁴ CMP and 8×10⁴ GMP (▪, n=8, 88% survival). *P<0.0001 ( versus◯) and **P=0.01 (▪ versus ◯). P=0.99 ( versus ▪).

FIG. 3A-3B. Analysis of the tissue distribution of CMP/GMP progeny andsurvival following A. fumigatus infection. (A) Flow cytometric analysisof tissue neutrophils (Mac-1⁺Gr-1⁺) on D+8 identified the presence ofdonor CMP/GMP-derived cells (CD45.1⁺CD45.2⁻). Although present in bothbone marrow and blood, the donor cells constituted the majority ofsplenic neutrophils in comparison to host cells (CD45.1⁺CD45.2⁺). (B)Kaplan-Meier plot of mice infected with 3-4×10⁶ cfu of A. fumigatusfollowing treatment with 5-FU only (open circles) or 5-FU+1×10³ CMP and2×10³ GMP (closed circles). The myeloid progenitors were infused 30hours after 5-FU administration and the mice were infected 8 dayspost-chemotherapy via intranasal instillation of A. fumigatus conidia.The group that had received the CMP/GMP infusion (n=41) had asignificantly higher survival rate than the group treated with 5-FU only(n=75) (56% and 33% respectively; P=0.019). Animals that succumbed toinfection at 2-4 days post-instillation showed clinical evidence ofdisease, whereas those that survived appeared healthy throughout theobservation period (30-60 days).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Myeloid function is enhanced in an individual through transplantation ofallogeneic or xenogeneic myeloid progenitor cells, where the cellscomprise one or more of: common myeloid progenitor cells (CMP); andcommitted myeloid progenitors: erythroid/megakaryocytic progenitor(MEP), granulocyte/monocyte progenitors (GMP); and megakaryocyteprogenitor (MKP). The myeloid progenitor cells may have one or moreClass I HLA mismatches relative to the recipient, and administration isoptionally performed without HLA testing of the recipient. The myeloidprogenitor cells may be obtained from bone marrow, mobilized peripheralblood, etc., and may be used fresh, frozen, after in vitro expansion,and the like. The myeloid progenitor cells may be administered incombination with committed cells in the lineage, which committed cellscan be immature cells, platelets, etc.

The methods of the invention find use in the prevention and treatment ofinfection, particularly infections that are at least partiallycontrolled by the innate immune system, which system includes dendriticcells, monocytes, macrophages, neutrophils, etc., and which pathogensmay include viruses, such as CMV; fungal pathogens such as Aspergillusspecies; bacteria such as Salmonella; and protozoan pathogens, such asToxoplasma. The methods of the invention also find use in alleviatinggastrointestinal mucositis, which is a serious complication ofimmunosuppression, for example resulting from chemotherapy,radiotherapy, and the like.

In some embodiments of the invention, infection is a fungal infection.Mycoses may be caused by primary pathogenic and opportunistic fungalpathogens. The primary pathogenic fungi are able to establish infectionin a normal host; whereas, opportunistic pathogens require a compromisedhost in order to establish infection (e.g., cancer, organtransplantation, surgery, and AIDS). Primary systemic fungal pathogensinclude Coccidioides immitis, Histoplasma capsulatum, Blastomycesdermatitidis, and Paracoccidioides brasiliensis. Opportunistic fungalpathogens include Cryptococcus neoformans, Candida spp., Aspergillusspp., Penicillium marneffei, the Zygomycetes, Trichosporon beigelii, andFusarium spp.

Myeloid Progenitors.

As used herein, the term refers generally to a class of mammalian cellsthat differentiate into cells of a myeloid lineage, and which lack thepotential to differentiate into lymphoid lineages, which class includesCMP, GMP, MEP and MKP cells, as defined below. These cells provide forphysiological functionality, such as protection from fungal infection,production of platelets and erythrocytes, etc., even in the presence ofone or more MHC Class I antigen differences between donor and host.Following transplantation, the presence of the donor cells and progenythereof is transient, where substantially all of the detectable donorcells are gone at about 4 weeks post-transplantation. These progenitorsare capable of rapid differentiation activity in vivo. CMP cells giverise to Gr-1+/Mac-1+ myelomonocytic cells and megakaryocytic colonies,as well as TER119+ erythroid cells in spleen and bone marrow. The GMPprogenitor population gives rise to Gr-1+/Mac-1+ cells; and the MEPprogenitor population to megakaryocytes and erythroid cells.

The myeloid progenitor subsets are isolated from any source ofhematopoietic progenitor cells, which may be fetal, neonatal, juvenileor adult, including bone marrow, spleen, liver, umbilical cord blood,peripheral blood, mobilized peripheral blood, yolk sac, etc. Forperipheral blood, progenitor cells are mobilized from the marrowcompartment into the peripheral bloodstream after treatment withchemotherapy; G-CSF or GM-CSF, or both. A number of single andcombination chemotherapeutic agents have been used to mobilize PBPCs. Inadministering these agents, a balance must be found in all cases betweeneffective PBPC mobilization and possible damage to the hematopoieticstem cell pool and overall patient tolerance. Paclitaxel has been foundto effectively mobilize PBPCs without damaging the stem cell pool. Areview of peripheral blood stem cells may be found in Shpall et al.(1997) Annu Rev Med 48:241-251, and the characterization of stem cellmobilization in Moog et al. (1998) Ann Hematol 77(4):143-7. As analternative source of cells, progenitor cells may be derived from the invitro culture of stem cells, e.g. embryonic stem cells, embryonic germcells, hematopoietic stem cells, etc.

The progenitor cells may be obtained from any mammalian species, e.g.equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats,hamster, primate, etc., particularly human. The tissue may be obtainedby biopsy or aphoresis from a live donor, or obtained from a dead ordying donor within about 48 hours of death, or freshly frozen tissue,tissue frozen within about 12 hours of death and maintained at belowabout −20° C., usually at about liquid nitrogen temperature (−180° C.)indefinitely.

For most purposes, the myeloid progenitor cells are isolated from otherhematopoietic cells, even when a defined cocktail of cells is ultimatelyprovided to a patient. The isolated population will usually have atleast about 75% cells of the selected phenotype, more usually at least85% cells of the selected phenotype; and may have 95% cells of theselected phenotype. For some purposes, the selected cells will comprisea single myeloid progenitor, e.g. CMP. For other purposes, the selectedcells will comprise two or more myeloid progenitors, e.g. CMP and GMP;CMP and MEP; CMP, MEP and MKP; CMP, GMP and MEP; and the like.

For selection of CMP in combination with GMP and MEP, the cells may beselected for the phenotype Thy-1⁻, IL-7Rα⁻, Lin⁻, and CD38⁺. Forselection of CMP in combination with GMP, the cells may further beselected for the IL-3Rα^(lo) phenotype. For selection of CMP incombination with MEP, the cells may be further selected for thephenotype CD45RA⁻.

The progenitor cells are usually separated from other cells, e.g.hematopoietic cells, on the basis of specific markers, which areidentified with affinity reagents, e.g. monoclonal antibodies. Themyeloid progenitor cells lack expression of lineage specific markers.For staining purposes a cocktail of binding reagents, herein designated“lin”, may be used. The lin panel will comprise binding reagents, e.g.antibodies and functional binding fragments thereof, ligands,peptidomimetics, etc., that recognize two or more of the lineagemarkers. A lin panel will generally include at least one markerexpressed on mature B cells, on mature T cells, on mature granulocytesand on mature macrophages. Markers suitable for use in a lineage panelare typically expressed on these mature cells, but are not present onmultiple lineages, or on stem and progenitor cells.

The myeloid progenitor cells are separated from a complex mixture ofcells by techniques that enrich for cells having the abovecharacteristics. For isolation of cells from tissue, an appropriatesolution may be used for dispersion or suspension. Such solution willgenerally be a balanced salt solution, e.g. normal saline, PBS, Hank'sbalanced salt solution, etc., conveniently supplemented with fetal calfserum or other naturally occurring factors, in conjunction with anacceptable buffer at low concentration, generally from 5-25 mM.Convenient buffers include HEPES, phosphate buffers, lactate buffers,etc.

Separation of the subject cell populations will then use affinityseparation to provide a substantially pure population. Techniques foraffinity separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or used in conjunction with amonoclonal antibody, e.g. complement and cytotoxins, and “panning” withantibody attached to a solid matrix, e.g. plate, or other convenienttechnique. Techniques providing accurate separation include fluorescenceactivated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. The cellsmay be selected against dead cells by employing dyes associated withdead cells (e.g. propidium iodide). Any technique may be employed whichis not unduly detrimental to the viability of the selected cells.

The affinity reagents may be specific receptors or ligands for the cellsurface molecules indicated above. In addition to antibody reagents,peptide-MHC antigen and T cell receptor pairs may be used; peptideligands and receptor; effector and receptor molecules, and the like.Antibodies and T cell receptors may be monoclonal or polyclonal, and maybe produced by transgenic animals, immunized animals, immortalized humanor animal B-cells, cells transfected with DNA vectors encoding theantibody or T cell receptor, etc. The details of the preparation ofantibodies and their suitability for use as specific binding members arewell-known to those skilled in the art. Conveniently, antibodies areconjugated with a label for use in separation. Labels include magneticbeads, which allow for direct separation, biotin, which can be removedwith avidin or streptavidin bound to a support, fluorochromes, which canbe used with a fluorescence activated cell sorter, or the like, to allowfor ease of separation of the particular cell type. Fluorochromes thatfind use include phycobiliproteins, e.g. phycoerythrin andallophycocyanins, fluorescein and Texas red. Frequently each antibody islabeled with a different fluorochrome, to permit independent sorting foreach marker.

The separated cells may be collected in any appropriate medium thatmaintains the viability of the cells, usually having a cushion of serumat the bottom of the collection tube. Various media are commerciallyavailable and may be used according to the nature of the cells,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequentlysupplemented with fetal calf serum.

The enriched cell population may be grown in vitro under various cultureconditions. Culture medium may be liquid or semi-solid, e.g. containingagar, methylcellulose, etc. The cell population may be convenientlysuspended in an appropriate nutrient medium, such as Iscove's modifiedDMEM or RPMI-1640, normally supplemented with fetal calf serum (about5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, andantibiotics, e.g. penicillin and streptomycin.

The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors. Specific growth factors that may be used inculturing the subject cells include steel factor (c-kit ligand), Flk-2ligand, IL-11, IL-3, GM-CSF, erythropoietin and thrombopoietin. Thespecific culture conditions are chosen to achieve a particular purpose,i.e. differentiation into erythroid of megakaryocyte populations,maintenance of progenitor cell activity, etc.

Genes may be introduced into the myeloid progenitor cells for a varietyof purposes, e.g. prevent HIV infection, replace genes having a loss offunction mutation, provide recognition of a particular antigen, suppressactivation of a particular antigen receptor, etc. Alternatively, vectorsare introduced that express antisense mRNA or ribozymes, therebyblocking expression of an undesired gene. Other methods of gene therapyare the introduction of drug resistance genes to enable normalprogenitor cells to have an advantage and be subject to selectivepressure, for example the multiple drug resistance gene (MDR), oranti-apoptosis genes, such as bcl-2. Various techniques known in the artmay be used to transfect the target cells, e.g. electroporation, calciumprecipitated DNA, fusion, transfection, lipofection and the like. Theparticular manner in which the DNA is introduced is not critical to thepractice of the invention.

Common Myeloid Progenitor (CMP).

A hematopoietic progenitor subset that can give rise to all lineages ofmyeloerythroid cells, but lacks the potential to differentiate intolymphoid lineages. The CMP cells may be identified and isolated by meansof cell surface markers. The CMP cells of both humans and mice stainnegatively for the markers Thy-1 (CD90), IL-7Rα (CD127); and with apanel of lineage markers, which lineage markers may include CD2; CD3;CD4; CD7; CD8; CD10; CD11 b; CD14; CD19; CD20; CD56; and glycophorin A(GPA) in humans and CD2; CD3; CD4; CD8; CD19; IgM; Ter110; Gr-1 in mice.The cells are CD34 positive, and CD38 positive. In humans, the CMP isalso characterized as IL-3Rα^(lo) CD45RA⁻. In the mouse the CMP areSca-1 negative, (Ly-6E and Ly-6A), c-kit high, and FcγR^(lo).

The developmental potential of CMPs can be demonstrated by in vitroculture in the presence of steel factor (SLF), flt-3 ligand (FL),interleukin (IL)-3, IL-11, GM-CSF, thrombopoietin (Tpo) anderythropoietin (Epo), where the CMP cells give rise to myeloerythroidcolonies including CFU-GEMMeg, burst-forming unit-erythroid (BFU-E),CFU-megakaryocytes (CFU-Meg), CFU-granulocyte/macrophage (CFU-GM),CFU-granulocyte (CFU-G) and CFU-macrophage (CFU-M).

Granulocyte/Monocyte Progenitor (GMP).

A hematopoietic progenitor subset that can give rise to monocytes andgranulocyte lineages, but lacks the potential to differentiate intolymphoid, erythroid and megakaryocytic lineages. The GMP cells of bothhumans and mice stain negatively for the markers Thy-1 (CD90), IL-7Rα(CD127); and with a panel of lineage markers, which lineage markers mayinclude CD2; CD3; CD4; CD7; CD8; CD10; CD11 b; CD14; CD19; CD20; CD56;and glycophorin A (GPA) in humans and CD2; CD3; CD4; CD8; CD19; IgM;Ter110; Gr-1 in mice. The cells are CD34 positive, and CD38 positive.The human cells are also IL-3Rα^(lo) CD45RA⁺, while the mouse isFcγR^(hi).

The developmental potential of GMPs can be demonstrated by in vitroculture in the presence of steel factor (SLF), flt-3 ligand (FL),interleukin (IL)-3, IL-11, GM-CSF, thrombopoietin (Tpo) anderythropoietin (Epo), where the CMP cells give rise to CFU-M, CFU-G, orCFU-GM colonies containing macrophages and/or granulocytes.

Megakaryocyte/Erythroid Progenitor (MEP).

A hematopoietic progenitor subset that can give rise to megakaryocyticand erythroid cells, but lacks the potential to differentiate intolymphoid, granulocytic and monocytic lineages. The MEP cells of bothhumans and mice stain negatively for the markers Thy-1 (CD90), IL-7Rα(CD127); and with a panel of lineage markers, which lineage markers mayinclude CD2; CD3; CD4; CD7; CD8; CD10; CD11b; CD14; CD19; CD20; CD56;and glycophorin A (GPA) in humans and CD2; CD3; CD4; CD8; CD19; IgM;Ter110; Gr-1 in mice. The human cells are CD34 positive, and CD38positive, and are also IL-3Rα⁻CD45RA⁻, while the mouse is FcγR^(lo)CD34⁻.

Megakaryocyte Progenitor (MKP).

A hematopoietic progenitor restricted to the megakaryocytic lineage. MKPcells express detectable levels of the markers CD41, CD9 and CD34.Optionally, the cells may be further selected for a lack of expressionof the markers Thy-1 (CD90), IL-7Rα (CD127); and/or with a lineage panelof markers. Other markers of interest include positive expression ofCD38 and c-kit (CD117). In one embodiment of the invention, a selectedset cells includes CMP, MEP and MKP cells, having the phenotype ofThy-1⁻, IL-7Rα⁻, lin⁻, CD34⁺, CD38⁺.

As used herein, “syngeneic” refers to a cells that are geneticallyessentially identical with the recipient or essentially all leukocytesof the recipient. Examples of syngeneic cells include cells derived fromthe recipient, also referred to in the art as “autologous cells”, aclone of the recipient, or an identical twin of the recipient. In animalmodels, congenic strains are substantially syngeneic, in that the cellsare identical at all loci except for the selected congenic marker.

As used herein, “allogeneic cells” refers to a cells derived from adonor that is non-syngeneic with the recipient or non-syngeneic with asubstantial proportion of the lymphocytes present in the recipient,where the donor is of the same species as the recipient or of the samespecies as substantially all of the lymphocytes of the recipient.Typically, non-clonal mammals of the same species are allogeneicrelative to each other.

As used herein, “xenogeneic” refers to a cells of a different speciesthan the recipient or of a different species than a substantialproportion of the lymphocytes present in the recipient.

HLA Mismatches and Typing.

Major histocompatibility complex antigens (also called human leukocyteantigens, HLA, or the H2 locus in the mouse) are protein moleculesexpressed on the surface of cells that confer a unique antigenicidentity to these cells. MHC/HLA antigens are target molecules that arerecognized by T-cells and natural killer (NK) cells as being derivedfrom the same source of hematopoietic reconstituting stem cells as theimmune effector cells (“self”) or as being derived from another sourceof hematopoietic reconstituting cells (“non-self”). Two main classes ofHLA antigens are recognised: HLA class I and HLA class II. HLA class Iantigens (A, B, and C in humans) render each cell recognisable as“self,” whereas HLA class II antigens (DR, DP, and DQ in humans) areinvolved in reactions between lymphocytes and antigen presenting cells.Both have been implicated in the rejection of transplanted organs.

An important aspect of the HLA gene system is its polymorphism. Eachgene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) existsin different alleles. HLA alleles are designated by numbers andsubscripts. For example, two unrelated individuals may carry class IHLA-B, genes B5, and Bw41, respectively. Allelic gene products differ inone or more amino acids in the α and/or β domain(s). Large panels ofspecific antibodies or nucleic acid reagents are used to type HLAhaplotypes of individuals, using leukocytes that express class I andclass II molecules.

The most important alleles for HLA typing are the six MHC Class Iproteins expressed by the host and donor: two alleles for each of HLA-A;HLA-B and HLA-C. The myeloid progenitor cells used in the methods of theinvention are typically mismatched for at least one Class I allele, andmay be mismatched for two, three, four, five or all of the Class Ialleles.

Any method known in the art may be optionally used to for typing themyeloid progenitor cells. For example, three main processes arecurrently used to perform HLA typing. The first is conventionalserological cytotoxicity method, where samples of lymphocytes (takenfrom blood or spleen) are added to Terasaki plates. These plates holdindividual wells that contain different specific antibodies (from eithermaternal sera or manufactured monoclonal antibodies). The best cells forclass II typing are B lymphocytes, and class I typing can be performedwith the remaining leucocytes. Magnetic beads are used to purify therequired cells from blood or spleen. If the HLA antigen and specificantibody bind, and complement is added, the cells in that well will bekilled. The pattern of wells showing this cell death allows thededuction of which combination of HLA antigens were present on theoriginal tissue cells.

Another method used for HLA typing is flow cytometry, particularly whenlooking for specific alleles. Leucocytes are added to detectable labeledmonoclonal antibodies specific for the HLA types of interest. The sampleis then analyzed by flow cytometry to determine which antibodies havebound to the cells.

DNA typing is increasingly being used for HLA typing. This processinvolves extracting the DNA from cells and amplifying the genes thatencode for the HLA peptides using polymerase chain reaction techniques.The genes may be matched with known HLA nucleotide sequences foundstored in several gene bank databases, including the IMGT/HLA database.

Cells between individuals may also differ at minor histocompatibilityantigens, or minor H antigens. Minor H antigens are peptides derivedfrom polymorphic proteins that are presented by the MHC molecules on thegraft. MHC class I molecules bind and present a selection of peptidesderived from proteins made in the cell, and if polymorphisms in theseproteins mean that different peptides are produced in different membersof a species, these can be recognized as minor H antigens. One set ofproteins that induce minor H responses is encoded on the male-specific Ychromosome. Responses induced by these proteins are known collectivelyas H-Y. The nature of the majority of minor H antigens, encoded byautosomal genes, is unknown, but one, HA-2, has been identified as apeptide derived from myosin.

An effective dose of one or more allogeneic myeloid progenitor cells isadministered to an individual in need of enhancement of myeloid orerythroid function. An effective dose of the allogeneic myeloidprogenitor cells varies within wide limits and will, of course be fittedto the individual requirements in each particular case. The number ofcells used will depend on the weight and condition of the recipient andother variables known to those of skill in the art. In general, suchamount is at least 10⁴ myeloid progenitor cells per kg of body weightand most generally need not be more than 3×10⁷ myeloid progenitorcells/kg, usually at least about 5×10⁵/kg, more usually at least about1×10⁶/kg; and not more than about 1×10⁷/kg, more usually not more thanabout 5×10⁶/kg. Where the cells are administered in combination with acytokine, the effective dose may be reduced, for example from about2×10⁵/kg to 5×10⁵/kg. The biological effectiveness of a dose may beempirically determined, e.g. in an animal model, and extrapolated forthe specific patient needs and size. End points of interest includeproduction of red blood cells sufficient to render a patient non-anemic;production of platelets sufficient to protect a patient fromcomplications due to bleeding; production of neutrophils sufficient toprotect a patient from fungal infection; and the like.

The cells can be administered by any route that is suitable for theparticular tissue or organ to be treated. The cells can be administeredsystemically, i.e., parenterally, by intravenous injection. The cellscan be suspended in an appropriate diluent, at a concentration of fromabout 0.5 to about 5×10⁶ cells/ml. As discussed above, the cells willgenerally be selected first to provide a purified population comprisingone or more myeloid progenitor subsets.

In a preferred embodiment, the myeloid progenitor cell preparation orcomposition is formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration tohuman beings. Typically, compositions for intravenous administration aresolutions in sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a local anesthetic to ameliorate any painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa cryopreserved concentrate in a hermetically sealed container such asan ampoule indicating the quantity of active agent. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical preparation of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

The present invention is particularly advantageous in that myeloidprogenitor cells may be used for a variety of treatments wherein thesource of the myeloid progenitor cells is other than the recipient andwithout requiring that such source be matched to the recipient.Moreover, such myeloid progenitor cells may be used without requiringchronic administration of immunosuppressants.

Patients suffering from various diseases and therapies may have adeficiency on one or more myeloid lineage cells, as a result of chronicdisease, infection, drug treatment, and the like. Such patients benefitfrom the administration of an effective dose of allogeneic myeloidprogenitor cells for enhancement of myeloid and/or erythroid function.The purified cells may be formulated with biologically active agents,such as antifungal agents, antibiotics, cytokines, etc., and maycomprise biological entities, such as red blood cells, platelets,hematopoietic stem cells, etc. The agents may be provided in a singleformulation with the cells, or may be separately administered.

For example, patients undergoing hematopoietic cell transplantation(HCT) receive myeloablative doses of chemo-radiation therapy that leadto depletion of hematopoietic stem cells (HSC), progenitor cells andmature cells, thus leading to a phase of treatment related pancytopenia.Embodiments of interest include the treatment of such patients inconjunction with myeloablative chemotherapy or radiation therapy, wherethe treatment comprises co-administration of hematopoietic stem cells incombination with CMP, and one or more of the cytokines such as G-CSF andGM-CSF. Myeloid progenitors may be given as additive or synergistictherapy with antimicrobials for the treatment or prevention ofinfections, including antibiotics and antifungal agents.

The use of G-CSF (neupogen) in the treatment of patients afterchemotherapy is known in the art, and it will be administered in dosesand regimens consistent with such practice. For example, neupogen dosesthat accelerate neutrophil production may range from about 1 μg/kg to100 μg/kg, and may delivered sub-cutaneously, intravenously, etc. Trialshave been reported for regimens including 4-8 μg/kg SC at days 4-17;5.75-46 μg/kg IV at days 4-17; 3.45-69 μg/kg IV at days 4-11; 23-69μg/kg IV at days 8-28; 11.5 μg/kg IV at days 2-9; 5.75 μg/kg IV at days10-12; and 5.45-17.25 μg/kg SC at days 6-19.

Neutropenia is a reduction in the blood neutrophil (granulocyte) count,often leading to increased susceptibility to bacterial and fungalinfections. Neutropenia may be classified by the neutrophil count[(total WBC)×(% neutrophils+bands)] and the relative risk of infection:mild (1000 to 1500/μL), moderate (500 to 1000/μL), or severe (<500/μL).Acute, severe neutropenia caused by impaired neutrophil production isoften life-threatening in immunocompromised patients. Cytotoxicchemotherapy induces neutropenia because of the high proliferative rateof neutrophil precursors and the rapid turnover of blood neutrophils.Impaired neutrophil production can also occur when leukemia, myeloma,lymphoma, or metastatic solid tumors infiltrate and replace the bonemarrow. Tumor-induced myelofibrosis may further extenuate neutropenia.Myelofibrosis can also occur from granulomatous infections, Gaucher'sdisease, and radiotherapy. Embodiments of interest include theadministration of CMP, optionally in combination with GMP, where thecells are provided in conjunction with G-CSF. Patients may also benefitfrom the administration of antifungal agents, e.g. amphotericin,AmBisome, Abelcet, Voriconazole, Caspofungin, Itraconazole, Fluconazole;and the like.

Anemia is a lower than normal number of red blood cells (erythrocytes)in the blood, usually measured by a decrease in the amount ofhemoglobin. Causes of anemia include B12 deficiency, iron deficiency,folate deficiency, hemolytic anemia, G-6-PD deficiency, idiopathicaplastic anemia, idiopathic autoimmune hemolytic anemia, immunehemolytic anemia, megaloblastic anemia, pernicious anemia, secondaryaplastic anemia, sickle cell anemia, etc. Embodiments of interest forthe treatment of anemia include administration of CMP and/or MEPoptionally in conjunction with erythropoietin.

Erythropoietin is a glycoprotein that stimulates red blood cellproduction. It is produced in the kidney and stimulates the division anddifferentiation of committed erythroid progenitors in the bone marrow.Anemia in cancer patients may be related to the disease itself or theeffect of concomitantly administered chemotherapeutic agents. The use ofEPO is known in the art, and the methods of the invention are consistentwith such practice. EPO has been shown to increase hematocrit anddecrease transfusion requirements after the first month of therapy, inanemic cancer patients undergoing chemotherapy. Endogenous baselineserum erythropoietin levels vary among patients. In general, patientswith lower baseline serum erythropoietin levels responded morevigorously than patients with higher baseline erythropoietin levels.Although no specific serum erythropoietin level can be stipulated abovewhich patients would be unlikely to respond to therapy, treatment ofpatients with grossly elevated serum erythropoietin levels (e.g., >200mUnits/mL) is not recommended. Intravenously administered EPO iseliminated at a rate consistent with first order kinetics with acirculating half-life ranging from approximately 4 to 13 hours. Withinthe therapeutic dose range, from about 1 to 250 U/kg, usually from about50-150 U/kg, detectable levels of plasma erythropoietin are maintainedfor at least 24 hours.

Thrombocytopenia is any disorder in which there are not enoughplatelets. This condition is sometimes associated with abnormalbleeding. Thrombocytopenia is often divided into three major causes oflow platelets: low production of platelets in the bone marrow, increasedbreakdown of platelets in the bloodstream, and increased breakdown ofplatelets in the spleen or liver. Disorders that involve low productionin the bone marrow include aplastic anemia, and cancer in the bonemarrow. Disorders that involve the breakdown of platelets include:immune thrombocytopenic purpura (ITP), drug-induced immunethrombocytopenia, drug-induced nonimmune thrombocytopenia, thromboticthrombocytopenic purpura, primary thrombocythemia, disseminatedintravascular coagulation (DIC), hypersplenism, etc. Embodiments ofinterest for the treatment of thrombocytopenia include administration ofCMP, optionally combined with MEP and/or MKP. The cell function may beenhanced by co-administration of thrombopoietin.

Thrombocytopenia following myelotoxic therapy is a common problem andwhen severe (<20 000/μl) can lead to severe morbidity and mortality.Thrombopoietin (TPO) is a naturally occurring glycosylated peptide whichstimulates the differentiation of bone marrow stem cells intomegakaryocyte progenitor cells, induces the expression of megakaryocytedifferentiation markers, promotes megakaryocyte proliferation,polyploidization and, ultimately, the formation of increased numbers ofplatelets in the circulation. TPO has now been produced by recombinanttechnology and has entered clinical trials. TPO will be administeredaccording to protocols as known in the art, for example, rhTPO may beadministered intravenously by bolus injection at doses ranging from 0.1to 10 μg/kg/day every one to 3 days. G-CSF may be concomitantlyadministered to promote myeloid recovery. (see Bone MarrowTransplantation (2001) 27, 261-268).

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

Example 1 The Ability of Allogeneic Committed Myeloid Progenitors toProtect Against Invasive Aspergillosis Following Myeloablative Treatmentis not MHC-Restricted

The results presented herein demonstrate that allogeneicco-transplantation of CMP/GMP protects mice against lethal invasiveaspergillosis, and, of great clinical significance, this protection isnot MHC restricted. The number of CMP/GMP transplanted and theMCH-antigen disparity between the host and donor did not change thefunctional outcome even though the quantitative reconstitution ofmyeloid cells was decreased in hosts with greater antigen disparity.These results further support our hypothesis that graft engineeringusing hematopoietic progenitor cell populations can be useful todecrease susceptibility to infections in patients undergoing HCT. Fullyallogeneic myeloid progenitor transplantations could improve upongranulocyte transfusions or administration of G-CSF to overcome periodsof neutropenia of any cause.

Methods

Animals.

CMP/GMP were isolated from the bone marrow of C57BL/Ka-Thy1.1.CD45.1(H2^(b)) mice and C57BL/6.CD45.2 (H2^(b)), BALB.B (H2^(b)), or Balb/c(H2^(d)) mice were used as hosts in congenic, allogeneic MHC-matched orMHC-mismatched transplantation experiments, respectively. HSC wereisolated from the bone marrow of C57BL/Ka-Thy1.1.CD45.2 (H2^(b)) orB10.D2.Thy1.1 (H2^(d)) mice. The use of these mouse strain combinationsallowed differentiation between the CMP/GMP-derived cells and the hostor HSC-derived cells. All mice were bred and maintained at the animalcare facility at Stanford University School of Medicine. Donor mice wereused at 6-8 weeks and hosts at 8-14 weeks of age.

Cell Sorting and Flow Cytometric Analysis.

To isolate c-Kit⁺Thy1.1^(lo)Lin⁻Sca-1⁺ HSC, whole bone marrow cells(WBMC) from femurs and tibiae were depleted of mature erythrocytes viaammonium chloride lysis and then stained with biotinylated ratanti-mouse c-Kit (3C11) followed by streptavidin immunomagnetic beads(Miltenyi Biotec, Auburn, Calif.) as described previously. c-Kit⁺ cellswere positively selected using an autoMACS cell separator (MiltenyiBiotec, Auburn, Calif.). Cells enriched for HSC were stained withPE-conjugated anti-CD3 (145-2C11, Pharmingen, San Diego, Calif.),anti-CD4 (GK1.5), anti-CD5 (53-7.8), anti-CD8 (53-6.7), anti-B220 (6B2),anti-Ter119, anti-Mac-1 (M1/70), and anti-Gr-1 (RB6-8C5) andFITC-conjugated anti-Thy1.1 (19XE5), Texas Red (TxR)-conjugatedanti-Sca-1 (E13-161) and allophycocyanin (APC)-conjugated anti-c-Kit(2B8).

To isolate Lin⁻Sca-1⁻c-Kit⁺CD34⁺FcγR^(lo) CMP andLin⁻Sca-1⁻c-Kit⁺CD34⁺FcγR^(hi) GMP, WBMC from C57BL/Ka-Thy1.1.CD45.1mice were depleted of mature erythrocytes via ammonium chloride lysisand then stained with biotinylated rat antibodies against the lineage(Lin) markers IL-7Rα-chain (A7R34), CD19 (1D3, Pharmingen, San Diego,Calif.), immunoglobulin M (IgM, Pharmingen, San Diego, Calif.), Thy1.1(19XE5), and unconjugated rat antibodies against Ter119, Gr-1 (RB6-8C5),and Cy5-PE conjugated rat anti-mouse CD45R/B220 (RA3-6B2, Pharmingen,San Diego, Calif.). The Lin⁺ cells were partially removed followingincubation with sheep anti-rat Dynabeads (Dynal, Oslo, Norway) andmagnetic depletion. Cells enriched for progenitors were then stainedwith CD45R/B220 (RA3-6B2, Pharmingen, San Diego, Calif.), streptavidinCy5-PE and goat anti-rat Cy5-PE (RED670, Gibco, Gaithersburg, Md.),TxR-conjugated anti-Sca-1 (E13-161), APC-conjugated anti-c-Kit (2B8),FITC-conjugated anti-CD34 (RAM34, Pharmingen, San Diego, Calif.), andPE-conjugated anti-CD16/32 (2.4G2, Pharmingen, San Diego, Calif.).

The reconstitution analysis of cellular subpopulations in blood, spleenand bone marrow samples was accomplished by depleting matureerythrocytes via ammonium chloride lysis and then staining withantibodies in various combinations including TxR or APC-conjugatedanti-CD45.1 (A20.1.7), TxR-conjugated anti-Mac-1 (M1/70), PE-conjugatedanti-Gr-1, FITC-conjugated anti-Ter119, PE-conjugated anti-MHC class II(I-A/I-E, M5/114.15.2, Pharmingen, San Diego, Calif.), APC-conjugatedanti-CD11c (HL3, Pharmingen, San Diego, Calif.). Antibody staining andwashing was performed using HBSS with penicillin/streptomycin (GibcoBRL, Grand Island, N.Y.) and 2% heat-inactivated fetal bovine serum FBS(SIGMA, St. Louis, Mo.). Cells were incubated 20-30 minutes at 4° C.,and washed twice. Dead cells were excluded by propidium iodide staining.

Cell sorting and analysis were performed on a modified dual laserfluorescence-activated cell sorter (FACSVantage, BD Biosciences, SanJose, Calif.) equipped with a 488-nm argon and a 595-nm dye laser madeavailable through the FACS shared user group at Stanford University.

Irradiation and Transplantation.

C57BL/6 hosts were irradiated with 9.5 Gy, BALB.B or Balb/c hosts with 8Gy, in 2 fractions, 3-4 hours apart, using a 200-kV x-ray machine(Philips RT250, Shelton, Conn.) and given antibiotic water (1.1 g/Lneomycin sulfate and 10⁶ U/L polymyxin B sulfate) after irradiation.Mice were anesthetized with Isoflurane (Abbott Laboratories, AbbottPark, Ill.) and cells were injected into the retro-orbital venous plexususing 0.5 cc insulin syringes with 28 Ga needles (Applied Scientific,South San Francisco, Calif.).

Cell Counts.

Blood samples were submitted to the diagnostic laboratory of theDepartment of Comparative Medicine at Stanford University where completeblood counts (CBC) including differential counts were performed on aCell DYN 3500 (Abbott Laboratories, Abbott Park, Ill.). Single cellsuspensions of splenocytes were stained with Turks solution and countedmanually using a hemocytometer.

Aspergillus fumigatus Infection.

A clinical isolate of Aspergillus fumigatus that had caused fatalsinusitis in a patient following allogeneic HCT was used in theseexperiments and prepared as described previously. On day 7post-transplantation, mice were infected intravenously (i.v.) vialateral tail vein with 100 conidia Aspergillus fumigatus in a totalvolume of 150 μl sterile normal saline. The infections were performed bya single individual who was blinded to the experimental groups. Micewere weighed daily and examined twice daily following infection.

Statistical Analysis.

For comparison of absolute cell counts, the rank sum test was performed.We used the log-rank test to compare groups in Kaplan Meier survivalanalysis.

Results

Rapid Myeloid Reconstitution from Allogeneic CMP/GMP.

In order to determine if MHC-antigen disparity influenced engraftment ofmyeloid progenitors, CMP/GMP-derived cells were quantitated in blood,spleen, and bone marrow on day 7 post-transplantation. Myeloidprogenitor engraftment after transplantation from congenic, MHC-matchedor MHC-mismatched donors was compared. Donor-host mouse strain pairingsare shown in Table 1.

TABLE 1 Experiment type CMP/GMP donor HSC donor Host CMP/GMP AntigenDisparity Function C57BL/6-Ly5.2 C57BL/Ka-Thy1-1 BALB.B MHC-matchedunrelated Function C57BL/6-Ly5.2 B10.D2.Thy1.1 Balb/c MHC-mismatchedQuantitative C57BL/6-Ly5.2 none C57BL/6 congenic engraftmentQuantitative C57BL/6-Ly5.2 none BALB.B MHC-matched unrelated engraftmentQuantitative C57BL/6-Ly5.2 none Balb/c MHC-mismatched engraftment

At this time-point, analysis of peripheral blood of mice in all groups(Table 1, groups 1 to 3) confirmed a significant peripheral bloodneutropenia with low white blood cell and low absolute neutrophil countsthat were not statistically significant between the three groupsalthough a tendency to lower counts with increasing antigen disparitywas observed (Table 2). Reliable FACS analysis from peripheral blood todetermine CMP/GMP-derived chimerism could not be performed because ofthe low number of white blood cells within the samples. Red blood cellcounts were within the normal range and were not different between thegroups.

TABLE 2 Reconstitution from CMP/GMP matched MHC- congenic unrelatedmismatched cell type unit mean SD mean SD mean SD Blood RBC ×10⁶//μl 7 39 0 8 1 Hematocrit % 31 12 40 2 39 3 WBC /μl 200 310 91 79 78 88 ANC /μl125 183 60 47 38 34 Spleen total CMP/GMP derived ×10⁵ 43.9 14.0 21.813.1 1.4 1.7 Total granulocytes 26.6 9.3 10.5 6.5 0.8 1.1CMP/GMP-derived granulocytes 26.6 9.3 10.5 6.5 0.8 1.1 Bone Marrow % %CMP/GMP-derived 42.9 16.8 35.3 16.1 20.1 10.8 % granulocytes 50.4 13.559 11.7 71.8 5.8 % granulocytes CMP/GMP derived 77.4 8.8 81.3 5.1 83.65.1

In contrast, high CMP/GMP-derived chimerism was detected in spleen andbone marrow (Table 2). In spleen and bone marrow, the majority ofCMP/GMP-derived cells were mature granulocytes as defined by theco-expression of the two markers Mac-1 and Gr-1. The remaining cellswere either erythroid cells (Ter119⁺) or cells of the dendritic celllineage (CD11c⁺MHC II⁺). With increasing degrees of antigen disparitybetween donor and host, the engraftment efficiency of the CMP/GMPpopulations decreased. When compared to congenic donor-host pairs,transplantation of allogeneic MHC-matched CMP/GMP was not significantlydifferent in absolute or relative number of CMP/GMP-derived cells. Therewas no significant difference in the percentage of CMP/GMP-derived cellsin the bone marrow of either allogeneic group when compared to congenicdonor-host pairings (FIG. 1C and Table 2).

CMP/GMP did not give rise to cells of the lymphoid lineage and theirmyeloid progeny could not be detected in blood, spleen or bone marrowlater than day 28 post-transplantation, confirming the purity of thesorted cell populations.

Co-Transplantation of Allogeneic CMP/GMP Protects Against LethalAspergillus fumigatus Infection.

In a separate series of experiments, designed to study the function ofMHC-matched allogeneic CMP/GMP, lethally irradiated mice wereco-transplanted with 10⁴ CMP, 2×10⁴ GMP and 500 HSC (Table 1 group 4).Mice that received grafts containing 500 HSC alone were used ascontrols. On day 7 post-transplantation, all mice were infected with 100conidia of A. fumigatus intravenously. Mice that received graftscontaining CMP/GMP were protected against lethal fungal challenge whencompared to mice that received grafts containing HSC alone (P=0.02, FIG.2A).

In order to determine if functional engraftment of myeloid progenitorswas MHC-restricted, MHC-mismatched allogeneic mice were used as CMP/GMPdonors. Using the same study design as was used for MHC-matcheddonor-host pairings, groups of lethally irradiated mice received graftscontaining the same number of CMP/GMP and HSC (Table 1 group 5). Micethat received grafts containing CMP/GMP from MHC-mismatched donors wereprotected against lethal invasive aspergillosis as compared to micetransplanted with HSC alone. Furthermore, increasing the dose oftransplanted CMP/GMP fourfold did not significantly improve survival(P<0.0001, FIG. 2B). Regardless of the differences in engraftmentpattern described above, protection against invasive aspergillosis wascomparable between all groups.

In the study presented here, we demonstrate that co-transplantation ofallogeneic CMP/GMP functionally improves immune reconstitution followingHSC transplantation. Inclusion of CMP/GMP in the graft acceleratesreconstitution of the tissue myeloid pool that protects mice againstinvasive aspergillosis early post-transplant. Of greatest clinicalimportance regarding the utility of such cellular therapy is theobservation that this protection is not MHC-restricted.

Patients undergoing HCT are at high risk for developing invasive fungalinfections—especially due to molds such as Aspergillus fumigatus—whichare associated with a high morbidity and mortality despite thedevelopment of new antifungal agents and supportive strategies such asgranulocyte transfusions or use of hematopoietic growth factors.Furthermore, establishing the diagnosis is often difficult involvingclinical, radiological, microbiological and histopathologicalexaminations. Therefore, strategies to reduce susceptibility toinfection are crucial for the outcome. Furthermore, it is of greatimportance to develop new therapeutic approaches to prevent theestablishment of bacterial or invasive fungal infections which would notbe dependent on antimicrobial therapy and the attendant risks ofinducing resistance. Although the risk factors for invasiveaspergillosis post-transplantation are not restricted to neutropenia,the biology and normal immune defenses against this mold make it aprototypic pathogen to determine whether or not functional myeloidreconstitution has been achieved.

The immune response to Aspergillus fumigatus involves cells of theinnate (macrophages and neutrophils, first lines of defense) and cellsof the adaptive immune system as second lines of defense (dendriticcells and T cells). CMP/GMP are progenitors committed to myeloid lineagedevelopment, have lost the self-renewal capacity and give rise to allcells of the myeloid lineage including granulocytes, monocytes,megakaryocytes, erythrocytes and dendritic cells. We previously reportedthat inclusion of CMP/GMP to the graft of pure HSC could accelerate andfunctionally improve the reconstitution of myeloid effector cells in thesetting of congenic donor-host pairings. We now demonstrate thatallogeneic CMP/GMP are similarly effective and function in anon-MHC-restricted fashion.

In our experiments, CMP/GMP engraftment was detected in the bone marrowand spleen as early as 4 days post-transplantation and was robust by day7 post-transplantation in all three donor-host strain combinationstested. Other studies assessing in vivo trafficking of purified HSC andmultipotent hematopoietic progenitor cell populations revealed bonemarrow as well as spleen to be the primary homing and proliferationsites of early hematopoietic cell populations following myeloablativetreatment. Although we did not perform real time imaging studies inliving animals, our results suggest that CMP/GMP rapidly home to theirbiological niches (bone marrow and spleen) and start proliferationsimilar to other early hematopoietic progenitor cell populations.Furthermore, these events seem to occur regardless of MHC disparity,albeit less frequently in MHC mismatched hosts.

The barrier to HSC engraftment associated with increasing degrees ofMHC-mismatch consists of host T and NK cells and has been welldocumented in mice, however the underlying mechanisms for theseobservations remain to be elucidated. HSC did not appear to be directlytargeted by NK cells. In order to achieve the same HSC function—measuredas radioprotection—10-60 fold higher numbers of purified HSC needed tobe transplanted into an MHC-mismatched mouse as compared to a congenicmouse. Therefore, we expected that transplantation of CMP/GMP intoallogeneic recipients would result in reduced engraftment when comparedto congenic transplantation. In our experiments, reconstitution fromCMP/GMP was quantitatively reduced with increasing degrees of antigendisparities. Qualitatively, CMP/GMP gave rise to all cells of themyeloid lineage with the same profile of distribution as in congenichosts. Interestingly, peripheral blood neutropenia was present in alldonor-host pairings tested, suggesting that CMP/GMP reconstitute thetissue myeloid pool before peripheral blood neutropenia is resolved.

Although transplantation of CMP/GMP from MHC-mismatched donors resultedin quantitatively fewer progenitor-derived cells in the spleen and bonemarrow, protection against invasive aspergillosis in MHC-matched andMHC-mismatched donor-host pairings was preserved. Thus, protection wasnot MHC-restricted indicating that CMP/GMP co-transplantation mainlyacts on improving the innate arm of immunity against A. fumigatus. Thisis consistent with results from our previous study where in vivodepletion of granulocytes using anti-Gr-1 antibody abrogated theprotective effect of CMP/GMP co-transplantation. Interestingly,increasing the number of transplanted CMP/GMP by 4-fold did not resultin better protection. As demonstrated in the setting of congenictransplantation, a relatively modest augmentation of the myeloid tissuepool was sufficient to protect mice against invasive aspergillosis andperipheral blood neutropenia did not correlate with effective antifungalimmune response.

In conclusion, these data show that the inclusion of CMP/GMP in thegraft protects mice against lethal invasive aspergillosis in the earlyphase post-transplantation. Although the engraftment is quantitativelyless in MHC-mismatched recipients, the function measured as protectionagainst invasive aspergillosis is conserved. These data demonstrate thatthe human counterpart of these progenitors, analogous to the infusion ofother blood products, can be collected from unrelated donors. This canprovide feasible therapy with a broader range of applicability asadjunctive treatment to decrease susceptibility to fungal infectionsresulting from neutrophil dysfunction or depletion.

Example 2 Single Infusion of Myeloid Progenitors Reduces Death fromAspergillus fumigatus Following Chemotherapy-Induced Neutropenia

Hematopoietic progenitors committed to the myeloid lineage, the commonmyeloid and granulocyte-monocyte progenitors (CMP/GMP) have been shownto protect against opportunistic pathogens following myeloablativeradiation; however, the efficacy of this approach has not been studiedin the setting of chemotherapy-induced neutropenia. In this mouse model,the infusion of CMP/GMP on the day after 5-fluorouracil (5-FU)administration (D+1) resulted in a significant increase in the number ofsplenic neutrophils by D+8 when compared to 5-FU only controls (P=0.02),the majority of which were CMP/GMP-derived (54%). Moreover, 19% and 28%of neutrophils in the blood and bone marrow, respectively, wereCMP/GMP-derived. Survival following intranasal challenge with the fungusAspergillus fumigatus was significantly higher in CMP/GMP-infused micethan the controls (56% and 33% respectively; P=0.019). Thus, a singleinfusion of CMP/GMP enhances tissue neutrophil content and increasessurvival against a lethal challenge with A. fumigatus in the setting ofchemotherapy-induced neutropenia.

Materials and Methods

Mice.

The F1 generation of the congenic strains of C57BL/Ka-Thy1.1 (CD45.2)and C57BL/Ka-Thy1.1 (CD45.1) mice was used as host mice. Donor myeloidcell progenitors were purified from the C57BL/Ka-Thy1.2 (CD45.1) strain.All mice were bred and maintained at the animal care facility atStanford University School of Medicine. Donor mice were used at 6-8weeks and recipients at 12-16 weeks.

5-FU Treatment.

A single dose of 150 mg/kg of 5-FU (American Pharmaceutical PartnersInc.; Schaumburg, Ill.) was administered intravenously 30 hours prior tomyeloid progenitor cell transplantation.

Myeloid Progenitor Cell Isolation and Transplantation.

Myeloid progenitor cells were purified from whole bone marrow asdescribed previously. Briefly, CMP and GMP were identified and isolatedby (1) excluding cells expressing: IL-7Rα-chain, CD19, IgM, Thy 1.1,Ter119, Gr-1, and CD45R/B220; and (2) the positive CMP/GMP markers,c-kit, Sca-1, CD34, and CD16/32. Cells were sorted using a modifiedfluorescence-activated cell sorter (FACSVantage, BD Biosciences; SanJose, Calif.). Host mice were anesthetized (Isoflurane; AbbottLaboratories; N. Chicago, Ill.) and cells were transplanted into theretro-orbital cavity. Mice were observed until fully recovered.

Preparation of A. fumigatus Conidia.

A clinical isolate of A. fumigatus that had caused fatal sinusitis in apatient following allogeneic bone marrow transplantation was used togenerate a conidial suspension as described previously. Suspensions weremaintained at 4° C.

The A. fumigatus conidia stock was diluted with sterile saline to aconcentration of 3-4×10⁶ cfu/20-30 μl. Mice were anesthetized withAvertin administered intraperitoneally (375 mg/kg). A. fumigatus conidiawere instilled intranasally with a precision microliter pipette (Rainin;Emeryville, Calif.).

Mice were killed when exhibiting clinical evidence of disease or on thespecified study days. Lungs were harvested for histologic examinationand/or cultured onto Sabouraud dextrose agar plates (BD Biosciences;Cockeysville, Md.) for fungal colonies.

Statistics.

The log-rank test was performed on the survival results and the rank sumtest was used to assess the significance of the absolute leukocytecounts.

Results and Discussion

To assess the duration of the neutropenia resulting from theadministration of 5-FU, peripheral blood leukocyte counts were measuredat weekly intervals. Eight days (D+8) following the administration of5-FU, the absolute leukocyte counts in both experimental groups (5-FUonly vs. 5-FU+CMP/GMP) was only slightly lower than the lower limit ofnormal; however, mice in both groups demonstrated profound neutropenia(Table 3). By D+13, total leukocyte and absolute neutrophil counts hadrebounded to above normal levels in both groups. In order to determinethe early trafficking of CMP and GMP progeny, immunophenotypic analysesof peripheral blood, spleen, and bone marrow cells was performed on D+8.By this time, mice that had received a single infusion of 1×10⁴ CMP and2×10⁴ GMP had a significantly higher number of splenic Mac-1⁺Gr-1⁺neutrophils than the 5-FU only group (Table 3, P=0.02). Moreover, 54% ofthese splenic Mac-1⁺Gr-1⁺ neutrophils were derived from the donorCMP/GMP population. Therefore, the contribution of the myeloidprogenitor populations more than doubled the total neutrophil pool inthe spleen compartment. Moreover, CMP/GMP-derived Mac-1⁺Gr-1⁺neutrophils were also identified in blood and bone marrow (FIG. 4A).These data demonstrate that CMP and GMP migrate and home tohematopoietic sites thus suggesting a functional engraftment in thismodel of chemotherapy-induced neutropenia.

TABLE 3 Absolute leukocyte counts following 5-FU treatment +/− myeloidprogenitor infusion 5-FU only 5-FU + CMP/GMP D + 8 D + 13 D + 8 D + 13BLOOD n = 15 n = 7 n = 9 n = 5 Absolute WBC 4.1 ± 0.4 12.0 ± 1.6 5.4 ±0.5 13.0 ± 0.6 counts (×10³/μl) (ref. range: 5.5-9.3) Absolute 0.002 ±0.007  3.6 ± 0.8 0.03 ± 0.01 2.6 ± 0. Neutrophil counts (×10³/μl) (ref.range: 0.825-2.604) SPLEEN n = 14 n = 10 Absolute 3.3 ± 0.4 ND 4.5 ± 1.1ND splenocytes (×10⁸) Total 0.6 ± 0.2 ND  1.2 ± 0.3* ND Mac-1⁺Gr-1⁺(×10⁶) CMP/GMP- 6.5 ± 2.0 ND derived (×10⁵) (54)^(†) Data is meanabsolute cell counts ± SEM. *significant difference from D + 8 counts (P= 0.02). ND, not determined. ^(†)indicates percentage of totalMac-1⁺Gr-1⁺ cells.

As potentially important immune effectors, tissue was analyzed for theircontent of dendritic cells, macrophages, and monocytes. Althoughhost-derived myeloid DCs (CD11c+Mac-1+) and plasmacytoid DCs(CD11c+B220+) were identified in the bone marrow and spleen, there wereno CMP/GMP-derived DCs in these tissues. There was no significantdifference in the total DCs between the two experimental groups in bonemarrow and spleen of both the myeloid and plasmacytoid DCs (p=0.28 vs.p=0.86 and p=0.26 vs. p=0.89, respectively). Moreover, neitherhost-derived nor progenitor-derived DCs were identified in theperipheral blood. Similarly, no CMP/GMP-derived monocytes/macrophages(Mac-1⁺Gr-1⁻) were identified in any compartment and analysis ofabsolute splenic counts of host-derived Mac-1⁺Gr-1⁻ cells confirmed thatthere was no significant difference between the two experimental groupsof 5-FU only vs. 5-FU+CMP/GMP (p=0.16).

The significance of these quantitative differences in Mac 1+Gr1+ cellswas confirmed by the ability of the infusion of CMP and GMP to protectagainst lethal challenge with A. fumigatus. Following intranasalinstillation of 3-4×10⁶ conidia, only 33% of the animals that hadreceived only 5-FU survived (n=75) compared with 56% of the groupinfused with CMP/GMP (n=41, P=0.019)(FIG. 4B). The majority of mice diedwithin 3 days post-infection. Cultures of organs confirmed the presenceof the A. fumigatus in the lungs. The absence of A. fumigatus in othertissues may be explained by the rapid morbidity and mortality due to theinfection. Of note, the CMP/GMP infusions were well tolerated andhistological evaluations did not reveal evidence of pulmonary injuryattributable to the infusion of cells such as a diffuse neutrophilicinfiltration. A neutrophilic infiltrate proximately associated with thepresence of hyphae was observed in the lung tissue of about half of themice that had received the single infusion of progenitors; in contrast,this infiltrate was not observed in the mice that had received 5-FUalone.

These data confirm that a single infusion of myeloid progenitor cellsreduces susceptibility to infection with A. fumigatus in a preclinicalmodel of chemotherapy-induced neutropenia. Moreover, the infusion ofCMP/GMP resulted in the ability to contain a rapidly invasive infectionwhen introduced in the biologically relevant route of inhalation. Wepreviously reported that following transplantation, the tissue and notthe peripheral blood content of myeloid effectors correlates witheffective innate immunity and the in vivo depletion of Mac-1⁺Gr-1⁺ cellsabrogated this protection. Thus, it is reasonable to conclude that thecontainment of infection results from appropriate homing to the site ofinfection.

In contrast to the efficacy of the single infusion of GMP/GMP, the useof mature granulocytes in clinical settings require repeated infusionsof freshly apheresed cells which is not only cumbersome but likelyengenders the production of anti-leukocyte antibodies. Additionally, instudies where the cells were characterized, up to 20% of the apheresedcells were not granulocytes and may have contributed to the beneficialor detrimental effects observed. As was observed following myeloablativeradiation, CMP/GMP infusion was well tolerated even in these mice with aless severe degree of immunosuppression. It is interesting to note thatthe use of G-CSF to mobilize leukocytes may result in a more rapid anddurable elevation of the peripheral blood leukocyte counts when comparedto counts following infusions of dexamethasone-mobilized cells. Whetheror not G-CSF administration influences the mobilization, proliferation,or function of the infused cells remains an area of investigation. G-CSFadministration following transplantation of CMP/GMP shortened the periodof susceptibility to lethal fungal infection following myeloablativeradiation.

As human myeloid progenitors have already been characterized in the bonemarrow and G-CSF-mobilized peripheral blood, these data support furtherinvestigation into the feasibility of cellular based therapies toreplenish depleted cell populations or serve as a bridge pendingrecovery of the hematopoietic system following chemotherapy orradiation. The methods also provide a means of ameliorating anemia andbleeding in patients suffering from a red blood cell and/or thrombocyticdeficiency.

Example 3 Role of Myeloid Progenitors in Protection AgainstRadiation-Induced Gut Injury

Gastrointestinal mucositis is a serious complication of intensivechemotherapy and radiotherapy (XRT), resulting in severe pain, impairednutrition, and an increased susceptibility to infection. As shown above,a single infusion of common myeloid and granulocyte-monocyte progenitors(CMP/GMP, c-kit+Sca1+CD34 CD16/32+Lin-Thy1-) protected mice againstlethal challenge with bacterial or fungal pathogens followingchemotherapy or myeloablative radiation. Additionally, this protectionis not HLA-restricted and correlated with the tissue myeloid content.

CMP/GMP infusion against radiation-induced injury in thegastrointestinal mucosa and liver is now shown. Following 950 cGyirradiation, Balb/c mice received infusions of one of the following: [1]hematopoietic stem cells (HSC, c-kit+Thy1loLin-Sca-1+); or [2] freshlyisolated 10,000 CMP/20,000 GMP+HSC. In all cases, the HSC wereMHC-matched (H2d), and CMP/GMP were MHC mismatched (H2b). A cohort ofmice served as XRT controls. Mice were sacrificed at d+6 or d+9post-transplantation.

Gross examination of the gastrointestinal tissue showed that mice in theXRT and HSC groups had severe hemorrhagic diarrhea in contrast to themild to moderate diarrhea observed in the group receiving freshlyisolated CMP/GMP or the expanded CMP/GMP. At both time points,histologic examination of gastrointestinal tissue from the XRT controlsrevealed sub-mucosal bleeding in gastric and intestinal tissue; necrosisand ulceration in the stomach; crowding of epithelial cells near thevilli in the small intestine; and ulcerative areas and small foci ofinflammation in the colon; and necrosis, portal tract damage, anddilated sinusoids/central veins in the liver.

In comparison, the histologic changes were significantly less dramaticin the stomachs, colons, and livers of the mice that had received eitherfreshly isolated or in vitro expanded CMP/GMP. In a parallel experiment,irradiated mice were transplanted with CMP/GMP from transgenic FVB.luc+mice that constitutively express firefly luciferase. Imaging of theliving mice and harvested organs at d+6 and d+9 post-transplantationindicated the presence of CMP/GMP in the stomach and intestinal tissue,correlating with sites of attenuated XRT damage in our histologicanalysis.

These findings support the clinical observation that repletion of themyeloid progenitor pool plays an important role in enhancing therecovery of radiation-injured mucosa.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the compoundsand methodologies that are described in the publications which might beused in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

What is claimed is:
 1. A method of providing myeloid function in apatient in need thereof, said method comprising: transplanting into saidpatient a biologically effective dose of allogeneic myeloid progenitorcells.